Method of treating a liver disease comprising administering ostreolysin orfunctionally related variant thereof

ABSTRACT

Disclosed are methods for treating, preventing and alleviating obesity, fatty liver syndrome, diabetes, one or more metabolic syndrome conditions or complications and/or cancer comprising administering an effective amount of ostreolysin, its functionally related variant, or an extract or mushroom extract comprising the same to subjects in need thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation application of U.S. patentapplication Ser. No. 16/671,465 filed Nov. 1, 2019 which is aContinuation application of U.S. patent application Ser. No. 15/127,022,filed Sep. 18, 2016, which is a National Phase of PCT InternationalPatent Application No. PCT/IL2015/050283, entitled OSTREOLYSIN,FUNCTIONALLY RELATED VARIANT THEREOF, EXTRACT COMPRISING OSTREOLYSIN ANDUSES THEREOF, filed Mar. 18, 2015 and which claims the benefit priorityof U.S. Provisional Patent Application Nos. 61/955,338 filed Mar. 19,2014, 61/955,874 filed Mar. 20, 2014 and 62/082,308 filed Nov. 20, 2014.All of the above referenced applications are incorporated herein byreference in their entirely.

FIELD OF INVENTION

The invention is directed to the use of ostreolysin (oly), functionallyrelated variant thereof, or extract or mushroom extract comprising olyfor treating, preventing, alleviating and/or reducing one or morecondition or complication associated with metabolic syndrome or fortreating or preventing obesity, fatty liver, diabetes and/or cancer.

BACKGROUND OF THE INVENTION

Obesity is at epidemic proportions with more than 300 million obesepeople world-wide and constantly rising. Obesity is not only a cosmeticproblem, but a life-threatening disease, reducing quality of life aswell as its longevity. Obesity increases the risk for many dreadfuldiseases, including type 2 diabetes, cardiovascular diseases and cancer,and is associated with insulin resistance, glucose intolerance anddyslipidemia. Therefore, there is an important need to understand themechanisms related to obesity and find ways to combat the deadly diseaseand its complications.

As a treatment perspective, finding an appropriate cure for obesity andrelated complications is extremely challenging due to the physiologicaland biochemical complexity of the disease. However, it is clear thatchanging energy homeostasis in favor of energy expenditure vs. energyintake will help in combating obesity. Therefore, identification ofcellular mechanisms able to increase whole body energy expenditure(“negative energy balance”) are advantageous as targets for obesitytherapy.

One option to increase energy expenditure is the uncoupling ofmitochondrial respiration in brown adipose tissue (BAT). In thisprocess, there is a regulated proton leak in the inner mitochondrialmembrane through uncoupling protein 1 (UCP1), resulting in thedissipation of energy as heat and increased fuel oxidation. Thissuggests that high amounts of active BAT would be beneficial in thebattle against obesity. Unfortunately, however, human adults are notconsidered to have sufficient amounts of BAT, in contrast to smallmammals and newborn humans. Therefore, finding ways to increase theactivity of BAT in adulthood would be beneficial in combating obesity byincreasing the oxidation of nutrients in the body.

The recent discovery of BAT in human adults and a better understandingof BAT development have encouraged the quest for new alternatives totreat obesity since obese individuals seem to have less brown adiposetissue mass/activity than do their lean counterparts. It is noteworthythat the activity of BAT is approximately fourfold higher in the leangroup than in the overweight/obese group.

From an anatomical point of view, brown fat cells are localized in twotypes of depots: discrete and diffuse. In humans, BAT of discretelocation is found in cervical-supraclavicular, perirenal/adrenal, andparavertebral regions around the major vessels and is probably presentto generate and distribute heat to maintain core temperature. Indistinction, diffuse brown fat cells exist in white adipose and appearin response to cold exposure or chronic catecholamine stimulation.

The metabolic syndrome, which comprises a cluster of metabolicabnormalities such as hyperlipidaemia, diabetes mellitus andhypertension, is a widespread and increasingly prevalent disease inwestern and industrialized countries.

Non-alcoholic fatty liver disease (NAFLD) is now recognized as thehepatic manifestation of the metabolic syndrome and is emerging as oneof the most common causes of chronic liver disease worldwide. NAFLDencompasses a wide disease spectrum ranging from simple hepaticsteatosis to steatohepatitis, advanced fibrosis and cirrhosis.Liver-related morbidity and mortality due to NAFLD are observed inpatients who have advanced fibrosis and cirrhosis. The mechanisms thataccelerate the progression of simple steatosis towards more debilitatingand advanced stages of NAFLD remain poorly understood, but generallyassume that it implies a two hit theory. Hepatic fat accumulationrepresents the ‘first hit’ of the disease and it has been suggested thatfat accumulation in hepatocytes is the hallmark of NAFLD and leaves themhighly vulnerable to a ‘second hit’, for example, injury by oxidativestress and inflammatory cytokines, such as TNF-α, monocytechemoattractant protein-1 (MCP-1) and other cytokines.

At present, no pharmacotherapy is available that can fully reverse orprevent steatohepatitis. Therefore, it is necessary to develop effectivetherapies for the treatment of NAFLD and the discovery of molecules orcompositions that may reduce the risk of NAFLD would be useful.

Colorectal cancer (CRC) is the second leading cause of death from canceramong adults in the United States as well as in Israel. Mortality ratesare in constant rise, which is why there is so much importance infinding factors that would reduce morbidity. As a treatment perspective,finding an appropriate cure for cancer and related complications isextremely challenging due to the complexity of the mechanisms involvedin this disease progression. Therefore, identification of cellularmechanisms involved and molecules able to suppress colon cancer cellproliferation and progression could be advantageous for cancertreatment.

Caveolin-1 (Cav-1) is the major protein component of caveolae,specialized lipid rafts that are recognized in electron micrographs as50-100 nm invaginations of the plasma membrane. Caveolae are foundprimarily in terminally differentiated mesenchymal cells includingadipocytes, endothelial cells and fibroblasts suggesting a possible roleof Cav-1 as a negative regulator of cell proliferation. Interestingly,Cav-1 has been implicated in the pathogenesis of oncogenic celltransformation, tumorigenesis and metastasis. Cells, including tumorcells, constantly face the decision of whether to survive andproliferate or to undergo programmed cell death (apoptosis). Therefore,identifying the pathways that are pro-apoptotic or anti-apoptotic hasimportant implications for controlling tumor cell growth.

SUMMARY OF THE INVENTION

In some embodiments of the invention, there is provided a method fortreating, preventing or reducing the severity of one or more conditionsor complications associated with metabolic syndrome in a subject inneed, comprising: administering a composition comprising an effectiveamount of oly, oly functionally related variant or a combination thereofto the subject in need.

According to some embodiments of the invention, the one or moreconditions or complications associated with metabolic syndrome isoverweight, obesity, lipodystrophy, fatty liver, NAFLD, NASH, chronicliver disease, cirrhosis or hepatocellular carcinoma.

According to some embodiments of the invention, the one or moreconditions or complications associated with metabolic syndrome is highblood/plasma glucose levels, glucose intolerance type II diabetes, highcholesterol levels, high lipid levels, or high triglyceride levels.

According to some embodiments of the invention, there is provided amethod for treating, preventing, decreasing or reducing cancer in asubject in need comprising administering a composition comprising aneffective amount of oly, oly functionally related variant or acombination thereof to the subject in need thereof.

According to some embodiments of the invention, the cancer is coloncancer.

According to some embodiments of the invention, the oly is a recombinantprotein.

According to some embodiments of the invention, the oly is produced in aprokaryotic cell.

According to some embodiments of the invention the prokaryotic cell is abacterial cell.

According to some embodiments of the invention there is provided aformulation comprising an effective amount of oly, oly functionallyrelated variant or a combination thereof for treating, preventing,decreasing or reducing cancer.

According to some embodiments of the invention, the cancer is a coloncancer.

According to some embodiments of the invention, there is provided aformulation comprising oly, oly functionally related variant orcombination thereof, for treating, preventing or reducing the severityof one or more conditions or complications associated with metabolicsyndrome.

According to some embodiments of the invention, the one or moreconditions or complications associated with metabolic syndrome isoverweight, obesity, fatty liver, NAFLD, NASH, chronic liver disease,cirrhosis or hepatocellular carcinoma.

According to some embodiments of the invention, the one or moreconditions or complication is associated with metabolic syndrome is highblood/plasma glucose levels, glucose intolerance type II diabetes, highcholesterol levels, high lipid levels, or high triglyceride levels.

According to some embodiments of the invention, the formulation is apharmaceutical formulation.

According to some embodiments of the invention, treating obesity oroverweight is associated with differentiating white adipocyte into brownadipocyte in a cell or inducing brown adipogenesis in a cell.

According to some embodiments of the invention, the gene expression ofbrown adipogenesis markers is increased following the treatmentsdescribed herein.

According to some embodiments of the invention there is provided amethod for treating, preventing or reducing the severity of at least oneor more conditions or complications associated with metabolic syndromein a subject in need comprising administering an effective amount of anextract comprising oly or mushroom extract comprising oly, or acomposition comprising an extract comprising oly or mushroom extractcomprising oly to the subject in need.

According to some embodiments of the invention, the one or morecondition or complication associated with metabolic syndrome isoverweight, obesity, lipodystrophy, fatty liver, NAFLD, NASH, chronicliver disease, cirrhosis or hepatocellular carcinoma.

According to some embodiments of the invention, the one or morecondition or complication associated with metabolic syndrome is highblood/plasma glucose levels, glucose intolerance type II diabetes, highcholesterol levels, high lipid levels, or high triglyceride levels.

According to some embodiments of the invention there is provided amethod for treating, preventing, decreasing or reducing cancer in asubject in need comprising administering an effective amount of anextract comprising oly or mushroom extract comprising oly or acomposition comprising an extract comprising oly or mushroom extractcomprising oly to the subject in need.

According to some embodiments of the invention, the cancer is coloncancer.

According to some embodiments of the invention there is provided anextract or mushroom extract comprising oly or a formulation comprisingan effective amount of an extract or mushroom extract comprising oly,for treating, preventing or reducing the severity of one or morecondition or complication associated with metabolic syndrome.

According to some embodiments of the invention there is provided anextract or mushroom extract comprising oly or a formulation comprisingan effective amount of an effective amount of an extract or mushroomextract comprising oly for treating, preventing, decreasing or reducingcancer.

According to some embodiments of the invention, the formulation is anutraceutical formulation, a food additive or a food supplement.

According to some embodiments of the invention, the mushroom extract isderived from Pleurotus mushroom.

According to some embodiments of the invention, the Pleurotus mushroomis Pleurotus Ostreatus mushroom or Pleurotus Pulmonarious mushroom.

According to some embodiments of the invention, the extract or theformulation described herein are in a form of a powder, solution,enteric coated table, suspension, emulsion, tablet, or capsule, anenteric coated tablet, gel, cream, ointment, foam, paste or injection.

According to some embodiments of the invention, the formulation is anutracetical composition or a dietary supplement and comprises a carriersuitable for food consumption.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 presents an SDS-PAGE image. Lyophilized oly purity was determinedby SDS-PAGE (15%) in the presence of a reducing agent. The respective 40to 5 μl correspond to 10, 5, 3.75, 2.5 and 1.25 μg/lane.

FIG. 2 presents gel filtration chromatography of oly on analyticalSuperdex 75 column, developed under non-denaturative conditions with 25mM Tris-HCl+300 corresponding to the expected molecular size of amonomer.

FIG. 3 is a graph presenting the cell viability of HCT116 and FHS 74 Intcell lines. Oly (62.5 μg/ml) was added for 24 hours and an MTT assay wasperformed.

FIG. 4 Western blot analysis of oly using the newly designed polyclonalantibody. Proteins extracted from P. Ostreatus fruiting bodies (50 μg,Fruiting Bodies (FB)) and the recombinant oly protein (2-4 μg) wereloaded on SDS-PAGE and transferred into nitrocellulose membrane. Thenewly designed polyclonal antibody against oly was used (1:2,500dilution).

FIGS. 5A and 5B are microscopic images demonstrating lipid dropletaccumulation in HIB-1B and 3T3-L1 cells. Particularly, FIG. 5A providesrepresentative light microscopic images of oly-induced lipidaccumulation in HIB-1B cells (10 μg/ml, 48 hours), wherein the upperimages represent the control cells while the lower images represent theoly treated cells. Further, the left side images represent the ×20magnification and the right side images are for the ×40 magnification.FIG. 5B provides representative confocal microscopic images of HIB-1Band 3T3-L1 cells, treated (oly-10 μg/ml, 48 hours) or not (control) andstained with Nile red, a lipid detector dye.

FIGS. 6A and 6B are graphs presenting the effects of oly (oly, 10 μg/ml,48 hours) on the gene expression of adipogenesis markers (6A) andspecific brown adipogenesis markers (6B). Gene expression was measuredby Real Time-PCR and normalized to beta-actin.

FIG. 7 is a graph presenting the oly-induced increase in the geneexpression of caveolin-1. Gene expression was measured by Real Time-PCRand normalized to beta-actin. Oly treatment was 10 μg/ml, 48 hours.

FIG. 8 is a graph representing the weight gain of mice submitted to thedifferent dietary conditions (High Fat Diet (HFD) and Low Fat Diet(LFD)) and different oly treatments. IPGTT was conducted on week 16.

FIG. 9 is a graph representing the weight at the sacrifice day of miceexposed to the different dietary conditions and oly treatments. * P<0.05from HFD.

FIGS. 10A and 10B are graphs representing the results of intraperitonealglucose tolerance test (IPGTT). (10A) Changes in blood glucose level offour experiment groups. (10B) Areas under the curve (AUC).

FIG. 11 is a graph representing the mouse food consumption.

FIG. 12 is a graph representing the weight of epididymal adipose tissueon day of sacrifice * P<0.001 from HFD.

FIGS. 13A-D are graphs representing the expression of UCP-1 (13A), Cidea(13B), PRDM16 (13C), perilipin A relative to B-actin (13D); FIG. 13E isa graph representing the expression of TNF-α relative to β actinexpression in visceral adipose tissue.

FIG. 14 is a graph representing the liver weights on day of sacrifice(P<0.05 from HFD group).

FIG. 15 is a graph representing the GOT levels on day of sacrifice(P<0.05 from HFD group).

FIG. 16 is a graph representing the GPT levels on day of sacrifice(P<0.05 from HFD group).

FIG. 17 is a graph representing the triglyceride levels on day ofsacrifice (P<0.05 from HFD group).

FIG. 18 is a graph representing the cholesterol levels on day ofsacrifice (P<0.05 from HFD group).

FIG. 19 A-D are images presenting the histological results of livers ofthe mice on the day of sacrifice; Control LF (19A), Control LF+oly(19B), HF (19C) and HF+oly (19D).

FIG. 20 is a graph representing the apoptosis assessment (BAX/BCL2) inthe livers of the mice on day of sacrifice (P<0.05).

FIGS. 21A-F show the cytotoxic activity of Ostreolysin (oly) towards HCT116 cells (21A, C and E) and HM7 clone #1 and clone #15 cells (21B, Dand F). Cells were grown overnight in Dulbecco's modified Eagle's mediumand treated with various concentrations of Ostreolysin, as indicated inthe Examples Section for 4 (21A and B), 8 (21C and D), 24 (21E and F)hours. Cell viability was estimated by the MTT assay. Viability (%) wasexpressed as the ratio between formazan absorbance at 550 nm of treatedcells at different time intervals and control cells at the beginning ofthe experiment. Each point represents the mean±SE from four independentexperiments performed in n=4 replicates. Error bars are notdistinguishable since they are smaller than symbol size.

FIG. 22 A and FIG. 22 B show fluorescence activated cell sortinganalysis of HCT116 cell line. Particularly, FIG. 22A presents cellstreated with Oly 125 μg/ml or FBE 0.01% (w/v), or left untreated ascontrol for 8 hours. Following incubation cells were harvested,permeabilized, stained with Propidium Iodide and analyzed. Results arerepresentative of one out of two independent experiments each performedin triplicates. Data were obtained from 15,000 HCT116 cells. Aspresented in FIG. 22B, the cell cycle was analyzed using WinMDI 2.9software of HCT116 cell line treated with Oly 125 μg/ml, FBE 0.01% (w/v)or left untreated as control and finally permeabilization and stainingas described above. All cell phases are represented as percentage. Datashown are the mean±SE of two independent experiments, each performed intriplicates. Data were obtained from 15,000 HCT116 cells.

FIGS. 23 A-C depict the effect of recombinant Oly on cleavage of PARP-1and BAX expression level in HCT116 cells. Cells were incubated for 8hours in the presence or absence of Oly or FBE (Fruiting BodiesExtract). Total cell lysates (for PARP-1 and BAX proteins) wereprocessed for western blot analysis as described in methods. FIG. 23A:Data shown are representative of one out of three independentexperiments, each performed in duplicates. FIG. 23B: Data shown arerepresentative of one out of four independent experiments, eachperformed in duplicates. Equal loading was confirmed by probing eachblot for β-actin. FIG. 23C: Quantification by densitometric analysis wasperformed using Gelpro32 analyzer software. Results are expressed asmean±SE (n=4) and statistical analysis indicated higher expression ofBAX in HCT116 cells treated with Oly versus untreated cells at P-value<0.05 (student's t-test).

FIG. 24 demonstrates that Ostreolysin penetrates the cell membrane andenters the cytosol. Representative immunofluorescence of HCT116 cellstreated for 8 hr, in non treated conditions (control), cells treatedwith Oly at a concentration of 125 μg/ml, cells treated with FBE at aconcentration of 0.01% (w/v) showing the presence of Ostreolysin insidethe cells, recognized by the anti-Oly antibody. Scale bar 20 μm.

FIG. 25 demonstrates that Ostreolysin induce Caveolin-1 reorganizationin lipid rafts. Representative immunofluorescence of HCT116 cellstreated for 8 hr in non treated conditions (control), cells treated withOly at a concentration of 125 μg/ml, cells treated with FBE at aconcentration of 0.01% (w/v) showing the clustering of Cav-1 on themembrane, recognized by the anti-Cav-1 antibody. Scale bar 20 μm. As canbe seen recombinant Oly enhances the expression of the lipid raft markerFlot-1 in HCT116 cells.

FIG. 26 demonstrates that Ostreolysin does not enhances Flotillin-1expression in lipid rafts. Therefore, Oly is specific for the raftprotein Cav-1. Representative immunofluorescence of HCT116 cells treatedfor 4 hr in non treated conditions, cells treated with Oly at aconcentration of 125 μg/ml and cells treated with FBE at a concentrationof 0.01% (w/v) showing the increased expression of Flot-1 on themembrane, recognized by the anti-Flot-1 antibody. Scale bar 20 μm.

FIG. 27 depicts the effect of Oly on MC38-derived colon cancer cellsimplanted in C57Bl mice. The cells were injected subcutaneously (2×10⁵cells per mouse) into the left hip. After 10 days of injection andfollowing appearance of tumor signs in some mice, the mice were treatedwith 1 mg/kg Oly, (3 times a week intraperitoneally). Control micereceived PBS three times a week intraperitoneally. Each bar representsthe standard error of the mean. N=8 mice; mice were sacrificed on day39. *=P<0.001.

FIG. 28 depicts the effect of Oly on MC38-derived colon cancer cellsimplanted in C57Bl mice. Tumors were excised after sacrifice andweighed. N=8; *=P<0.001.

FIG. 29 is a Western blot image showing the concentration of oly inextract preparations of Pleurotus Ostreatus after freezing in liquidnitrogen (Method 1, lanes 1 and 3) and powdered Pleurotus Ostreatusafter freezing in −20° C. (Method 2, lanes 2 and 4).

FIGS. 30A-D show control (30A), oly 10 μg/ml (30B), and extractpreparations of Pleurotus Ostreatus after freezing in liquid nitrogen(30C), and powdered Pleurotus Ostreatus after freezing in −20° C. (30D).

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the invention are directed to a method for preventing,alleviating and/or reducing one or more condition or complicationassociated with metabolic syndrome or for treating or preventingobesity, lipodystrophy, fatty liver, diabetes and/or cancer, the methodcomprising administering to a subject a therapeutically effective amountof oly, oly functionally related variant, or a composition comprisingoly or oly functionally related variant. In some embodiments, thesubject is administered with an extract or mushroom extract comprisingoly or a composition comprising an extract or mushroom extractcomprising oly. In some embodiments of the invention, the mushroomextract is an extract from Pleurotus mushroom. In some embodiments ofthe invention, the Pleurotus mushroom is derived from PleurotusOstreatus mushroom. In some embodiments of the invention, the Pleurotusmushroom is derived from Pleurotus Pulmonarious mushroom. It is notedthat throughout the application, unless specifically mentionedotherwise, the term “treating” is meant to include “preventing”,“reducing the effects/severity of the condition” “slowing down theprogression of the condition”, reducing/eliminating at least oneundesired side effect of the condition” and the like. The term“preventing” means that the compounds of the present invention areuseful when administered to a patient who has not been diagnosed aspossibly having the disease at the time of administration, but who maybe expected to develop the disease or be at increased risk for thedisease. The oly or its functionally related variant or an extractcomprising oly or a composition comprising oly or its functionallyrelated variant or an extract comprising oly of the invention will slowthe development of disease symptoms, delay the onset of the disease, orprevent the individual from developing the disease at all. Preventingalso includes administration of the compounds of the invention to thoseindividuals thought to be predisposed to the disease due to age,familial history, genetic or chromosomal abnormalities, and/or due tothe presence of one or more biological markers for the disease, such asa known genetic mutation.

The use of oly or its functionally related variant or an extractcomprising oly can prevent, retard, or improve various metabolicdiseases or disorders (e.g., obesity, metabolic syndrome, insulinresistance, diabetes, (including type 2 diabetes), and dyslipidemia) andtheir clinical complications such as acute myocardial infarction (“heartattack”) and aortic stenosis and other cardiovascular complications suchas but not limited to atherosclerosis; chronic kidney disease(particularly in view of diabetes impact on kidney vasculature);arterial calcification; valvular calcification, including but notlimited to aortic or mitral calcification; valvular stenosis, includingbut not limited to, aortic or mitral valve stenosis; acute myocardialinfarction; restenosis after coronary intervention; accelerated tissuedamage or delayed healing after coronary intervention, including but notlimited to: valve implantation (including bioprosthetic valveimplantation); stent implantation; implantation of engineered tissues,allograft, homograft (including but not limited to, Ross procedure),bioprosthesis tissues, Dacron grafts or any synthetic or bioprostheticconduit; heart transplantation; arterial or vein graft implantation(including but not limited to, saphenous vein bypass grafts andhemodialysis AV shunts); stroke; and heart failure; failure of veingrafts for coronary bypass surgery; diabetic nephropathy; vasculitis;retinopathy; erectile dysfunction; and non-cardiovascular complicationssuch as, but not limited to, pancreatitis; nonalcoholic fatty liverdisease; neuroinflammation; cognitive impairment; cancer.

In treating or preventing the diseases or conditions mentioned in theapplication, the compounds or extracts of the invention are administeredin a therapeutically effective amount. The therapeutically effectiveamount will vary depending on the particular compound used and the routeof administration, as is known to those skilled in the art.

Further, the amount of a composition to be administered will, of course,depend on the subject being treated, the severity of the affliction, themanner of administration, the judgment of the prescribing physician, andall other relevant factors. Determination of the exact dose to beadministered is conducted by methods known to a person of skill in theart.

In some embodiments of the invention, the amount of oly or thefunctional variant thereof in a single treatment is calculated to bebetween about 0.10-10 mg/kg body weight (BW) per day. In someembodiments of the invention, an amount of oly between about 0.3-1.0mg/kg is used per day. In some embodiments, an amount of between about0.5-0.8 mg/kg is used per day.

In case of an extract or a mushroom extract that comprises oly, theamount of the dry extract to be administered may be calculated accordingto the amount of the oly therein. In some embodiments, between about20-200 mg freeze dried powdered Pleurotus or Pleurotus Ostreatus/Kg bodyweight (BW) is used per day. In some embodiments, between about 20-60 mgfreeze dried powdered Pleurotus Ostreatus/Kg BW is used per day. In someembodiments, between about 40-50 mg freeze dried powdered PleurotusOstreatus/Kg BW is used per day.

Typically it is contemplated that treatment with oly, its fuctionallyrelated variant or extract comprising oly or a mushroom extractcomprising oly would be given at least once per day, typically once,twice, three times or four times per day with the doses given at equalintervals throughout the day and night in order to maintain a constantpresence of the drug in order to induce sufficient effect. However, theskilled artisan will be aware that a treatment schedule can be optimizedfor any given patient, and that administration of compound may occurless frequently than once per day.

The treatment may be carried out for as long a period as necessary.Typically it is contemplated that treatment would be continuedindefinitely while the disease state persists, although discontinuationmight be indicated if the compounds no longer produce a beneficialeffect. The treating physician will know how to increase, decrease, orinterrupt treatment based on patient response.

In some embodiments of the invention, the extract or the mushroomextract comprises oly in a concentration of at least 0.001 mg/g extractpowder. In some embodiments of the invention, the extract or themushroom extract comprises oly in a concentration of at least 0.005 mg/gpowder. In some embodiments of the invention, the extract or themushroom extract comprises oly in a concentration of at least 0.01 mg/gpowder. In some embodiments of the invention, the extract or themushroom extract comprises oly in a concentration of at least 0.02,0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1 mg/g extract powder ormore.

It is further understood that the utilized oly or its functionallyrelated variant can be formulated or administered together withadditional active ingredients as required to treat the condition of thepatient.

Ostreolysin (Oly) is a protein found in the Pleurotus Ostreatus mushroom(also known as oyster mushroom and Yarden mushrooms) and in PleurotusPulmonarious. Oly a 15-kDa cytolytic protein expressed during fruitingbodies formation that may interact with cholesterol enriched domains.

The present invention is based on the surprising effects of oly andextracts comprising oly on in-vivo and in-vitro models and variousparameters related metabolic syndrome and/or on fatty liver, obesity,over-weight, cancer and diabetes as well as the level of GOT, GPT,triglycerides and cholesterol.

As clearly seen in the Examples section, low amounts of recombinant oly,as well as extracts comprising oly, showed a significant effect on lipiddroplets accumulation in cells (FIG. 5), gene expression of adipogenesismarkers (FIG. 6), weight gain (FIG. 8), IPGTT (FIG. 10), weight ofepididimal adipose tissue (FIG. 12), liver weight and expression ofgenes (FIGS. 14 and 13, respectively) and other parameters like GOT,GPT, triglycerides and cholesterol.

As used herein, “Metabolic syndrome” or “syndrome X” is defined here onthe basis of NCEP ATP III criteria, which are the presence of three ormore of the following factors: 1) increased waist circumference (>102 cm[>40 in] for men, >88 cm [>35 in] for women); 2) elevated triglycerides(>150 mg/dl); 3) low HDL cholesterol (<40 mg/dl in men, <50 mg/dl inwomen); 4) non-optimal blood pressure (>130 mmHg systolic or mmHgdiastolic); and 5) impaired fasting glucose (>110 mg/dl). It is to beunderstood that the method of the invention is intended for treatingmetabolic syndrome as defined herein as well as one or more of any oneof the conditions of metabolic syndrome, separately or in combinationdefined herein or the complications described above.

As used herein, the term “fatty liver” refers to a condition where fataccumulates excessively in liver cells due to the disorder of lipidmetabolism. It may cause various diseases such as angina, myocardialinfarction, stroke, arteriosclerosis and pancreatitis.

“Impaired glucose tolerance” is defined here on the basis of AmericanDiabetes Association criteria. Impaired glucose tolerance is two-hour75-g oral glucose tolerance test values of 140 to 199 mg per dL (7.8 to11.0 mmol/l).

“Impaired fasting glucose” is defined here on the basis of AmericanDiabetes Association criteria. Impaired fasting glucose is defined asfasting plasma glucose values of 100 to 125 mg per dL (5.6 to 6.9mmol/l).

“Diabetes Mellitus” generally refers to fasting plasma glucose values ofequal or greater than 126 mg/dL (7.0 mmol/l).

“Insulin resistance” is defined here as a fasting blood insulin levelgreater than 20 mcU/mL.

“New onset diabetes” (usually defined on the basis of a fasting bloodglucose concentration of 7.0 mmol/l or more) in an individual.

“Hyperglycemia” is a fasting blood glucose concentration of 7.0 mmol/lor greater.

Fatty liver, also known as fatty liver disease (FLD), is a reversiblecondition. Large vacuoles of triglyceride fat accumulate in liver cellsvia the process of steatosis (the abnormal retention of lipids within acell). Fatty liver can be considered a single disease that occursworldwide in those with excessive alcohol intake and the obese (with orwithout effects of insulin resistance). The condition is also associatedwith other diseases that influence fat metabolism. When this process offat metabolism is disrupted, the fat can accumulate in the liver inexcessive amounts, thus resulting in a fatty liver. Both alcoholic FLDfrom nonalcoholic FLD, have similar symptoms and are characterized bymicrovesicular and macrovesicular fatty changes at different stages.

Accumulation of fat may also be accompanied by a progressiveinflammation of the liver (hepatitis), called steatohepatitis. Hepaticsteatosis” refers to a process describing the abnormal retention oflipids within a hepatocyle. Fatty liver may be termed alcoholicsteatosis or nonalcoholic fatty liver disease (NAFLD), depending on thelacohol consumption of the subject and the more severe forms asalcoholic steatohepatitis (part of alcoholic liver disease) andNon-alcoholic steatohepatitis (NASH).

For adults, “over-weight” and “obesity” ranges are determined by usingweight and height to calculate a number called the “body mass index”(BMI). BMI is used because, for most people, it correlates with theiramount of body fat. An adult who has a BMI between 25 and 29.9 isconsidered overweight. A BMI of 30 or higher is considered obese a BMIof 30 or higher is considered obese. All of these conditions ordisorders are improved or treated by oly, oly related variant or acomposition comprising an extract comprising oly, which may be mushroomextract and in some embodiments Pleurotus extracts.

As used herein, the term “oly” or “Ostreolysin” refers to the nativeOstreolysin protein or to the recombinant Ostreolysin protein,comprising the amino acid sequence set forth in:

(SEQ ID NO: 1) AYAQWVIIIIHNVGSQDVKIKNLKASWGKLHADGDKDAEVSASNYEGKIIKPDEKLQINACGRSDAAEGTTGTFDLVDPADGDKQVRHFYWDCPWGSKTNTWTVSGSNTKWMIEYSGQNLDSGALGTITVDTLKKGN.

As used herein, the terms “fragment” or “oly fragment” refer to anyamino acid sequence portion of oly.

As used herein, the terms “oly derivatives” or “oly analogs” refer tooly or oly fragment comprising at least one altered amino acid residueby an amino acid substitution, addition, deletion, or chemicalmodification, as compared with the sequence of oly or oly fragment. Olyderivatives include amino acid substitutions and/or additions withnaturally occurring amino acid, with non-naturally occurring amino acid,with any chemically modified amino acid and with amino acid with anyavailable molecular architecture.

As used herein, the phrase “oly functionally related variant” refers toany fragment, derivative or analog of oly or any combination thereof,having the same or enhanced functional activity of oly described herein.

In some embodiments of the invention, the oly functionally relatedvariant have at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequenceidentity to the amino acid sequence set forth in SEQ ID NO. 1 or to theamino acid sequence of the oly protein.

In some embodiments of the invention, oly, oly fragments, olyderivatives, oly analogs or oly functionally related variant areisolated proteins. In some embodiments of the invention, oly, olyfragments, oly derivatives, oly analogs or oly functionally relatedvariant are embedded in or connected to a carrier or moleculararchitecture of any type, size and atomic composition.

The improvement of a metabolically related parameter, metabolicassociated disease and/or pathological condition related to metabolism,refers to one or more of the following (separately or in combination ofone or more): reduction in weight, increase in energy consumption(possibly by increase in brown fat adipocytes production), improvementin liver related parameters, including liver mass, liver function (forexample by improvement in the activity of liver enzymes such as GOT andGPT) and number of fatty droplets, improvement in clinical parametersassociated with Non-alcoholic fatty liver disease (NAFLD), improvementin glucose related parameters, such as, blood/plasma glucose levels,improvement of glucose intolerance, and improvement in one or moreparameters related to cholesterol, lipid, peptide, leptin andtriglyceride levels.

It is known that NAFLD is the leading cause of chronic liver disease,wherein 20-30% of NAFLD patient progress to develop non-alcoholicsteatohepatitis (NASH), which in turn can lead to cirrhosis,hepatocellular carcinoma and increased mortality, type II diabetes,hyperlipimedimia, hypercholesterolemia and diseases wherein a clinicalbeneficial effect is manifested by improvement of liver function or atleast one related liver parameter. Thus, those conditions may also betreated by the method and the preparations of the present invention.

According to some embodiments, oly, its functionally related variant, orextract or mushroom extract comprising oly, its functionally relatedvariant or a composition comprising oly or oly functionally relatedvariant, or extract or mushroom extract comprising oly or compositionextract or mushroom extract comprising oly may be used in the treatmentof obesity, diabetes and/or complications thereof, possibly viapromoting brown adipocyte differentiation and increasing energyexpenditure. According to further embodiments, oly, its functionallyrelated variant, or extract or mushroom extract comprising oly or acomposition comprising oly, its functionally related variant, or extractor mushroom extract comprising oly may be used to improve liverfunction, reduce liver mass. According to some embodiments of theinvention, oly, its functionally related variant, or extract or mushroomextract comprising oly or a composition comprising oly, its functionallyrelated variant, or extract or mushroom extract comprising oly may beused to improve glucose intolerance in animals fed with a high fat diet.According to further embodiments, oly, its functionally related variant,or extract or mushroom extract comprising oly or a compositioncomprising oly, its functionally related variant, or extract or mushroomextract comprising oly are used to increase the metabolism of alreadyfully differentiated adipocytes.

According to some embodiments of the invention there is provided amethod of differentiating white adipocyte into brown adipocyte in a cellor inducing brown adipogenesis comprising contacting the cell with oly,oly functionally related variant or any combination thereof or anextract comprising oly. The method may be used for in-vitro tests, forexample, without limitation for assessing synergistic effects between ofoly and other active materials.

Further embodiments of the invention are directed to a method forincreasing metabolic rate by contacting fat cells with an effectiveamount of oly, its functionally related variant, or extract or mushroomextract comprising oly or a composition comprising oly, its functionallyrelated variant, or extract or mushroom extract comprising oly.According to some embodiments, the fat cells are brown, white or both.According to some embodiments, the higher metabolic rate can be causedby increased respiration and/or nutrient oxidation.

Further embodiments of the invention are directed to a method forenhancement of production of brown fat adipocytes the method comprisingcontacting a precursor of brown fat adipocytes with an effective amountof oly, its functionally related variant, or extract or mushroom extractcomprising oly or a composition comprising oly, its functionally relatedvariant, or extract or mushroom extract comprising oly.

Further embodiments of the invention are directed to methods of treatingcancer comprising the administration of an effective amount of oly, itsfunctionally related variant, or extract or mushroom extract comprisingoly or a composition comprising oly, its functionally related variant,or extract or mushroom extract comprising oly. According to someembodiments, the treated cancer is colon cancer. According to otherembodiments of the invention, the cancer is brain cancer, oropharyngealcancer, nasopharyngeal cancer, renal cancer, biliary cancer, prostaticcancer, pheochromocytoma, pancreatic islet cell cancer, Li-Fraumenitumors, thyroid cancer, parathyroid cancer, pituitary tumors, adrenalgland tumors, osteogenic sarcoma tumors, multiple neuroendrcine type Iand type II tumors, breast cancer, lung cancer, head & neck cancer,prostate cancer, esophageal cancer, tracheal cancer, skin cancer braincancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer,ovarian cancer, uterine cancer, cervical cancer, testicular cancer,colon cancer, rectal cancer or skin cancer. As used herein the term“cancer” refers to the presence of cells possessing characteristicstypical of cancer-causing cells, for example, uncontrolledproliferation, loss of specialized functions, immortality, significantmetastatic potential, significant increase in anti-apoptotic activity,rapid growth and proliferation rate, and certain characteristicmorphology and cellular markers. Typically, the cancer cells are in theform of a tumor; existing locally within an animal, or circulating inthe blood stream as independent cells, for example, leukemic cells. Thetreatment of cancer includes any reduction in the proliferation ofcancer cells, as well as the prevention of the cancer from occurring,recurring, decreasing the growth rate, cessation of growth, tumorshrinkage, slowing the progression, decreasing metastasis, increasingsurvival rate, increasing quality of life of the cancer patients, andthe like. The reduction of the proliferation of cancer cells may beachieved by decreased growth rates, cytostatic effects, cytotoxiceffect, apoptotic effects, or any combination thereof.

In some embodiments, oly, its functionally related variant, or extractor mushroom extract comprising oly induce cell arrest and/or apoptosis.In some embodiments, oly, its functionally related variant, or extractor mushroom extract comprising oly induce cell arrest or apoptosis in acancerous cell. In some embodiments, oly, its functionally relatedvariant, or extract or mushroom extract comprising oly inhibitmetastasis. In some embodiments, oly, its functionally related variant,or extract or mushroom extract comprising oly inhibit tumor growth. Insome embodiments, oly, its functionally related variant, or extract ormushroom extract comprising oly inhibit angiogenesis. In someembodiments, oly, its functionally related variant, or extract ormushroom extract comprising oly inhibit cell cycle. In some embodiments,oly, its functionally related variant, or extract or mushroom extractcomprising oly inhibit aberrant cell cycle.

According to some embodiments, the oly utilized according to the presentinvention is native oly, produced by mushrooms. According to someembodiments, the oly utilized according to the present invention is arecombinant protein. According to further embodiments, the oly isproduced in a prokaryotic cell. According to some embodiments, the olyis produced in bacterial cells. According to some embodiments, the olyproduced is not hemolytic. According to some embodiments, the bacterialcells are E. coli.

Some embodiments are directed to a pharmaceutical composition or anutraceutical composition comprising as an active ingredient atherapeutically effective amount of oly, its functionally relatedvariant, or extract or mushroom extract comprising oly, and apharmaceutically or nutraceutically acceptable carrier. The presentinvention further provides a dietary supplement comprising oly or olyfunctionally related variant or an extract or a mushroom extractcomprising oly. Pharmaceutically acceptable salts of the activeingredient (oly or a functionally related variant thereof) may also beused according to some embodiments. Pharmaceutically acceptable saltsinclude those salts formed with free amino groups such as salts derivedfrom non-toxic inorganic or organic acids such as hydrochloric,phosphoric, acetic, oxalic, tartaric acids, and the like, and thosesalts formed with free carboxyl groups such as salts derived fromnon-toxic inorganic or organic bases such as sodium, potassium,ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, and the like.

The pharmaceutically or the nutraceuticlly composition comprising oly,its functionally related variant, or extract or mushroom extractcomprising oly may comprise one or more additional active ingredientwhich is required to treat metabolic syndrome, or one or more of themetabolic syndrome conditions or complications described herein, as wellas one or more of obesity, fatty liver, diabetes, cancer and high levelof cholesterol and/or triglyceride. It is noted that the additionalactive ingredient may have synergistic effect with oly, oly, itsfunctionally related variant, or with the extract or mushroom extractcomprising oly.

The oly, its functionally related variant, or extract or mushroomextract comprising oly of the invention may also be prescribed to betaken in combination with other drugs used to treat obesity,lipodystrophy, appetite control in obesity, metabolic syndrome (asherein defined) as well as one or more of its conditions and/orcomplications (as herein defined) and lipodystrophy-related infertilityor obesity, fatty liver, diabetes, cancer and high level of cholesteroland or triglyceride. When used in such combinations, the oly or itsfunctionally related variant or extracts comprising oly and conventionaldrugs may be administered simultaneously, by the same or differentroutes, or at different times during treatment. The dose of theconventional drug selected will depend on the particular compound beingused and the route and frequency of administration.

The invention also provides a pharmaceutical or nutraceutical pack orkit comprising one or more containers filled with one or more of theingredients of the pharmaceutical or the nutraceutical compositions ofthe invention. Optionally associated with such container(s) is a noticein the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration.

The term “pharmaceutically acceptable” means suitable for administrationto a subject, e.g., a human. For example, the term “pharmaceuticallyacceptable” can mean approved by a regulatory agency of the Federal or astate government or listed in the U. S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans. The term “carrier” refers to a diluent, adjuvant, excipient, orvehicle with which the therapeutic compound is administered. Suchpharmaceutical carriers can be sterile liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents such as acetates, citrates or phosphates. Antibacterial agentssuch as benzyl alcohol or methyl parabens; antioxidants such as ascorbicacid or sodium bisulfate; and agents for the adjustment of tonicity suchas sodium chloride or dextrose are also envisioned.

The pharmaceutical or nutraceutical compositions or the dietarysupplements can take the form of solutions, suspensions, emulsions,tablets, or capsules. The pharmaceutical compositions can also take theform of powders, gels, creams, ointments, foams, pastes,sustained-release formulations and the like. The compositions can beformulated as a suppository, with traditional binders and carriers suchas triglycerides, microcrystalline cellulose, gum tragacanth or gelatin.Oral formulation can include standard carriers such as pharmaceuticalgrades of mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, cellulose, magnesium carbonate, etc. Examples of suitablepharmaceutical carriers are described in: Remington's PharmaceuticalSciences” by E. W. Martin, the contents of which are hereby incorporatedby reference herein. Such compositions will contain a therapeuticallyeffective amount of oly, its functionally related variant, e.g., in asubstantially purified form, together with a suitable amount of carrierso as to provide the form for proper administration to the subject. Insome embodiments of the invention, the nutraceutical or the dietarysupplement may be added to a food such as a chewing gum, a dairy productor the like. In some embodiments of the invention, the nutraceutical orthe dietary supplement may be added to liquid, such as water, milk orjuice,

The amount of oly, its functionally related variant, or extract ormushroom extract comprising the same, which will be effective in thetreatment of a particular disorder or condition will depend on thenature of the disorder or condition, and can be determined by standardclinical techniques known to a person skilled in the art. In addition,in-vitro assays may optionally be employed to help identify optimaldosage ranges. The precise dose to be employed in the formulation willalso depend on the route of administration, and the nature of thedisease or disorder, and should be decided according to the judgment ofthe practitioner and each patient's circumstances. Effective doses canbe extrapolated from dose-response curves derived from in-vitro orin-vivo animal model test bioassays or systems.

The route of administration of the pharmaceutical composition willdepend on the patient, and/or disease or condition to be treated.Suitable routes of administration include, but are not limited to,parenteral administration, e.g., intradermal, intravenous,intramuscular, intralesional, subcutaneous, intrathecal,intraperitoneal, and any other mode of administration as known in theart. According to some embodiments, the composition is administered viaoral, transdermal, rectal, vaginal, topical, nasal, inhalation andocular modes of treatment. Pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer.

For oral applications, the pharmaceutical composition may be in the formof tablets or capsules, which can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose; a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate; or aglidant such as colloidal silicon dioxide. When the dosage unit form isa capsule, it can contain, in addition to the ingredients of the abovetype, a liquid carrier such as fatty oil. In addition, dosage unit formscan contain various other materials which modify the physical form ofthe dosage unit, for example, coatings of sugar, shellac, or otherenteric agents. The tablets of the invention can further be film coated.

The term “precursor of brown fat adipocytes” refers to any cell that candifferentiate directly, or through intermediate cell types into brownfat adipocytes, including, for example, stem cells, mesenchymal stemcells, myogenic precursor, brown pre-adipocyte and white pre-adipocyte.

The terms “analog” and “derivative” refer to a peptide comprising atleast one altered amino acid residue by an amino acid substitution,addition, deletion, or chemical modification, as compared with thenative peptide. Peptide derivatives particularly include amino acidsubstitutions and/or additions with naturally occurring amino acidresidues, and chemical modifications such as, for example, enzymaticmodifications, typically present in nature. Peptide analogs particularlyinclude amino acid substitutions and/or additions with non-natural aminoacid residues, and chemical modifications which do not occur in nature.

The present invention encompasses the use of peptides/proteins of whichat least one amino acid has been chemically modified. Chemicalmodifications of amino acid residues include, but are not limited to,amidation, methylation, acetylation, glycosylation, oxidation,reduction, myristylation, sulfation, acylation, ADP-ribosylation,cyclization, hydroxylation, iodination, derivatization byprotecting/blocking groups, or any other derivatization method known inthe art. Such alterations, which do not destroy, but may improve thebiological activity of the oly.

In one embodiment, as used herein the terms “fragment” and “peptide” maybe used interchangeably having all the same meanings and qualities.

In one embodiment, as used herein the terms “peptide” and “protein” maybe used interchangeably having all the same meanings and qualities.

The derivatives, analogs, precursors, and fragments according to theprinciples of the present invention can also include side chain bondmodifications, including but not limited to —CH2-NH—, —CH2-S—, —CH2−S═O,O═C—NH—, —CH2-O—, —CH2-CH2-, S═C—NH—, and —CH═CH—, and backbonemodifications such as modified peptide bonds. Peptide bonds (—CO—NH—)within the peptide can be substituted, for example, by N-methylatedbonds (—N(CH3)-CO—); ester bonds (—C(R)H—C—O—O—C(R)H—N); ketomethylenebonds (—CO—CH2-); α-aza bonds (—NH—N(R)—CO—), wherein R is any alkylgroup, e.g., methyl; carba bonds (˜CH2-NH—); hydroxyethylene bonds(—CH(OH)—CH2-); thioamide bonds (—CS—NH); olefinic double bonds(—CH═CH—); and peptide derivatives (—N(R)—CH2-CO—), wherein R is the“normal” side chain, naturally presented on the carbon atom. Thesemodifications can occur at one or more of the bonds along the peptidechain and even at several (e.g., 2-3) at the same time.

The present invention also encompasses peptide/protein derivatives andanalogs in which free amino groups have been derivatized to form aminehydrochlorides, p-toluene sulfonylamino groups, carbobenzoxyaminogroups, t-butyloxycarbonylamino groups, chloroacetylamino groups orformylamino groups. Free carboxyl groups may be derivatized to form, forexample, salts, methyl and ethyl esters or other types of esters orhydrazides, and amides. The imidazole nitrogen of histidine can bederivatized to form N-im-benzylhistidine.

Also included are those peptide/protein derivatives, which contain oneor more naturally occurring amino acid derivatives of the twentystandard amino acid residues. For example: 4-hydroxyproline can besubstituted for proline; 5-hydroxylysine can be substituted for lysine;3-methylhistidine can be substituted for histidine; homoserine can besubstituted or serine; and ornithine can be substituted for lysine. Thepeptide analogs can also contain non-natural amino acids. Examples ofnon-natural amino acids include, but are not limited to, sarcosine(Sar), norleucine, ornithine, citrulline, diaminobutyric acid,homoserine, isopropyl Lys, 3-(2′-naphtyl)-Ala, nicotinyl Lys, aminoisobutyric acid, and 3-(3′-pyridyl-Ala).

Furthermore, the peptide/protein analogs can contain other derivatizedamino acid residues including, but not limited to, methylated aminoacids, N-benzylated amino acids, O-benzylated amino acids, N-acetylatedamino acids, 0-acetylated amino acids, carbobenzoxy-substituted aminoacids and the like. Specific examples include, but are not limited to,methyl-Ala (MeAla), MeTyr, MeArg, MeGlu, MeVal, MeHis, N-acetyl-Lys,O-acetyl-Lys, carbobenzoxy-Lys, Tyr-O-Benzyl, Glu-O-Benzyl, Benzyl-His,Arg-Tosyl, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, and the like.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant”, including where the alterationresults in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. Guidance concerning whichamino acid changes are likely to be phenotypically silent can also befound in Bowie et al., 1990, Science 247: 1306 1310. Such conservativelymodified variants are in addition to and do not exclude polymorphicvariants, interspecies homologs, and alleles. Typical conservativesubstitutions include but are not limited to: 1) Alanine (A), Glycine(G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine(L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)). Amino acids canbe substituted based upon properties associated with side chains, forexample, amino acids with polar side chains may be substituted, forexample, Serine (S) and Threonine (T); amino acids based on theelectrical charge of a side chains, for example, Arginine (R) andHistidine (H); and amino acids that have hydrophobic side chains, forexample, Valine (V) and Leucine (L). As indicated, changes are typicallyof a minor nature, such as conservative amino acid substitutions that donot significantly affect the folding or activity of the protein.

As used herein, in one embodiment, the term “amino acid derivative”refers to a group derivable from a naturally or non-naturally occurringamino acid, as described and exemplified herein. Amino acid derivativesare apparent to those of skill in the art and include, but are notlimited to, ester, amino alcohol, amino aldehyde, amino lactone, andN-methyl derivatives of naturally and non-naturally occurring aminoacids. In an embodiment, an amino acid derivative is provided as asubstituent of a compound described herein, wherein the substituent is—NH-G(Sc)—C(O)-Q or —OC(O)G(Sc)-Q, wherein Q is —SR, —NRR or alkoxyl, Ris hydrogen or alkyl, Sc is a side chain of a naturally occurring ornon-naturally occurring amino acid and G is C1-C2 alkyl. In certainembodiments, G is Ci alkyl and Sc is selected from the group consistingof hydrogen, alkyl, heteroalkyl, arylalkyl and heteroarylalkyl.

In some embodiments of the invention, the amino acids of theprotein/peptide are of L or D stereoisomers or combination thereof.

As used herein, in one embodiment, the terms “peptide” or “protein” or“fragment” may be derived from a natural biological source, synthesized,or produced by recombinant technology. It may be generated in anymanner, including by chemical synthesis. One or more of the amino acidsmay be modified, for example, by the addition of a chemical entity suchas a carbohydrate group, a phosphate group, a farnesyl group, anisofamesyt group, a fatty acid group, an acyl group (e.g., acetylgroup), a linker for conjugation, functionalization, or other knownprotecting/blocking groups.

In one embodiment, peptide/protein of the present invention are purifiedusing a variety of standard protein purification techniques, such as,but not limited to, affinity chromatography, ion exchangechromatography, filtration, electrophoresis, hydrophobic interactionchromatography, gel filtration chromatography, reverse phasechromatography, concanavalin A chromatography, chromatofocusing anddifferential solubilization.

In one embodiment, to facilitate recovery, the expressed coding sequencecan be engineered to encode the peptide/protein of the present inventionand fused cleavable moiety. In one embodiment, a fusion protein can bedesigned so that the peptide/protein can be readily isolated by affinitychromatography; e.g., by immobilization on a column specific for thecleavable moiety. In one embodiment, a cleavage site is engineeredbetween the peptide and the cleavable moiety and the peptide can bereleased from the chromatographic column by treatment with anappropriate enzyme or agent that specifically cleaves the fusion proteinat this site [e.g., see Booth et al., Immunol. Lett. 19:65-70 (1988);and Gardella et al., J. Biol. Chem. 265:15854-15859 (1990)].

In one embodiment, the protein/peptide of the present invention can alsobe synthesized using in-vitro expression systems. In one embodiment,in-vitro synthesis methods are well known in the art and the componentsof the system are commercially available.

In one embodiment, production of a protein/peptide of this invention isusing recombinant DNA technology. A “recombinant” peptide, or proteinrefers to a peptide, or protein produced by recombinant DNA techniques;i.e., produced from cells transformed by an exogenous DNA constructencoding the desired peptide or protein.

In one embodiment, a peptide of this invention comprises at least 5amino acids. In another embodiment, a peptide comprises at least 10amino acids. In another embodiment, a peptide comprises at least 20amino acids. In another embodiment, a peptide comprises at least 25amino acids. In other embodiments, a peptide comprises at least 30 aminoacids or at least 50 amino acids or 75 amino acids, or 100 amino acids,or 125 amino acids, or 150 amino acids, or 200 amino acids, or 250 aminoacids or 300 amino acids or 350 amino acids or 400 amino acids. In oneembodiment, a peptide of this invention consists essentially of at least5 amino acids. In another embodiment, a peptide consists essentially ofat least 10 amino acids. In other embodiments, a peptide consistsessentially of at least 30 amino acids or at least 50 amino acids or 75amino acids, or 100 amino acids, or 125 amino acids, or 150 amino acids,or 200 amino acids, or 250 amino acids or 300 amino acids or 350 aminoacids or 400 amino acids. In one embodiment, a peptide of this inventionconsists of at least 5 amino acids. In another embodiment, a peptideconsists of at least 10 amino acids. In other embodiments, a peptideconsists of at least 30 amino acids or at least 50 amino acids or 75amino acids, or 100 amino acids, or 125 amino acids, or 150 amino acids,or 200 amino acids, or 250 amino acids or 300 amino acids or 350 aminoacids or 400 amino acids.

Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted forsynthetic non-natural acid such as TIC, naphthylelanine (Nol),ring-methylated derivatives of Phe, halogenated derivatives of Phe oro-methyl-Tyr.

As used herein, in one embodiment the term “amino acid” refers tonaturally occurring and synthetic α, β γ or δ amino acids, and includesbut is not limited to, amino acids found in proteins, i.e. glycine,alanine, valine, leucine, isoleucine, methionine, phenylalanine,tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine,glutamine, aspartate, glutamate, lysine, arginine and histidine. Incertain embodiments, the amino acid is in the L-configuration.Alternatively, the amino acid can be a derivative of alanyl, valinyl,leucinyl, isoleuccinyl, prolinyl, phenylalaninyl, tryptophanyl,methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl,asparaginyl, glutaminyl, aspartoyl, glutaroyl, lysinyl, argininyl,histidinyl, β-alanyl, β-valinyl, β-leucinyl, β-isoleuccinyl, β-prolinyl,β-phenylalaninyl, β-tryptophanyl, β-methioninyl, β-glycinyl, β-serinyl,β-threoninyl, β-cysteinyl, β-tyrosinyl, β-asparaginyl, β-glutaminyl,β-aspartoyl, β-glutaroyl, β-lysinyl, β-argininyl or β-histidinyl. Asused herein, in one embodiment the phrase “Conservatively modifiedvariants” applies to both amino acid and nucleic acid sequences. “Aminoacid variants” refers to amino acid sequences. With respect toparticular nucleic acid sequences, conservatively modified variantsrefers to those nucleic acids which encode identical or essentiallyidentical amino acid sequences, or where the nucleic acid does notencode an amino acid sequence, to essentially identical or associated(e.g., naturally contiguous) sequences. Because of the degeneracy of thegenetic code, a large number of functionally identical nucleic acidsencode most proteins. For instance, the codons GCA, GCC, GCG and GCU allencode the amino acid alanine. Thus, at every position where an alanineis specified by a codon, the codon can be altered to another of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations”, which are onespecies of conservatively modified variations. Every nucleic acidsequence herein which encodes a polypeptide also describes silentvariations of the nucleic acid. One of skill will recognize that incertain contexts each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, silent variations of a nucleic acidwhich encodes a polypeptide is implicit in a described sequence withrespect to the expression product.

In some embodiments, the invention further envisages inclusion of theoly or its functionally related variant or a fused protein thereof in acomplex where it is attached to proteinaceous (e.g., heterologous aminoacid sequence) or non-proteinaceous moieties (e.g., PEG), each of whichbeing capable of prolonging the half-life of the composition while incirculation.

Such a molecule is highly stable (resistant to in-vivo proteaolyticactivity, probably due to steric hindrance conferred by thenon-proteinaceous moiety) and may be produced using common solid phasesynthesis. Further recombinant techniques may still be used, whereby therecombinant peptide product is subjected to in-vitro modification (e.g.,PEGylation as further described hereinbelow).

The phrase “non-proteinaceous moiety” as used herein refers to amolecule not including peptide bonded amino acids that is attached tothe above-described IL-31 amino acid sequence. According to someembodiments the non-proteinaceous moiety may be a polymer or aco-polymer (synthetic or natural). Non-limiting examples of thenon-proteinaceous moiety of the present invention include polyethyleneglycol (PEG) or derivative thereof, Polyvinyl pyrrolidone (PVP), divinylether and maleic anhydride copolymer (DIVEMA); polysialic acid (PSA)and/or poly(styrene comaleic anhydride) (SMA). Additionally, complexeswhich can protect oly or its functionally related variant from theenvironment and thus keep its stability may be used, including, forexample, liposomes or micelles containing oly or its functionallyrelated variant or a fused protein comprising thereof are also includedin the invention.

According to some embodiments of the invention, the oly or itsfunctionally related variant or the fused protein comprising oly or itsfunctionally related variant of the invention are attached to anon-proteinaceous moiety, which may act as a sustained-release enhancingagent. Exemplary sustained-release enhancing agents include, but are notlimited to hyaluronic acid (HA), alginic acid (AA), polyhydroxyethylmethacrylate (Poly-HEMA), glyme and polyisopropylacrylamide.

Attaching the amino acid sequence component of the oly or itsfunctionally related variant or the fused protein comprising thereof ofthe invention to other non-amino acid agents may be by covalent linkingor by non-covalent complexion, for example, by complexion to ahydrophobic polymer, which can be degraded or cleaved producing acompound capable of sustained release; by entrapping the amino acid partof the oly or its functionally related variant or the fused proteincomprising thereof in liposomes or micelles to produce a complexcomprising the oly or its functionally related variant or the fusedprotein comprising the same. The association may be by the entrapment ofthe amino acid sequence within the other component (liposome, micelle)or the impregnation of the amino acid sequence within a polymer toproduce the final peptide of the invention.

In some embodiments, the PEG derivative is N-hydroxysuccinimide (NHS)esters of PEG carboxylic acids, succinimidyl ester of carboxymethylatedPEG (SCM-PEG), benzotriazole carbonate derivatives of PEG, glycidylethers of PEG, PEG p-nitrophenyl carbonates (PEG-NPC, such as methoxyPEG-NPC), PEG aldehydes, PEG-orthopyridyl-disulfide,carbonyldimidazol-activated PEGs, PEG-thiol, PEG-maleimide.PEG-maleimide, PEG-vinylsulfone (VS), PEG-acrylate (AC) orPEG-orthopyridyl disulfide may be also used.

The non-proteinaceous moiety may be attached to the oly or itsfunctionally related variant amino acid sequence in any chosen position,provided that the therapeutic activity of oly or its functionallyrelated variant is retained.

In some embodiments of the invention, there is provided a fused proteinthat comprises oly or its functionally related variant and a proteinthat stabilizes oly or its functionally related variant or protect it inthe blood stream or at the tissue. In some embodiments of the invention,there is provided a fused protein that comprises oly or its functionallyrelated variant and IgG. The IgG may any subclasses or isotypes thereof,e.g., IgG1, IgG2, IgG3, IgG4.

In some embodiments of the invention, the oly or its functional relatedvariant and IgG and the IgG and/or any other protein that may be usedfor extending the half-life of oly or its functional related variant andIgG in the serum are linked by a linker. In some embodiments of theinvention, the linker is a sequence of between 2-20 amino acids.

In some embodiments of the invention, the linker is a sequence ofbetween 4-12 amino acids which form a cleavage site for enzymes such asMMP9/2, trypsin, PSA, cathepsins, kallikreins, serine proteases,caspases and others. Additional possible cleavage sites are presented inCHOI et al., “Protease-Activated Drug Development”, Theranostics, Vol.2(2), pp. 156-178 (found in http://www.thno.org/v02p0156.pdf). In someembodiments, the linker is between 6-8 amino acids and in someembodiments includes a cleavage site for enzymes such as MMP9/2,trypsin, PSA, cathepsins, kallikreins, serine proteases, caspases and/orothers.

In some embodiments, the linker that comprise a cleavage site ofMMP-9/2, cathepsin, trypsin, kallikreins, serine proteases, caspases orany other cleaving enzyme that can be added between oly or itsfunctional related variant and IgG. In some embodiments, the inventionprovides a fused protein comprising oly or its functional relatedvariant and IgG.

Furthermore, the present invention encompasses nucleic acids encodingthe fusion proteins described herein. The invention further encompassesnucleic acids encoding oly or oly functionally related variant. Inaddition, vectors comprising these nucleic acids and cells transformedwith such vectors are encompassed by the present invention.

In some embodiments of the invention an extract comprising oly may beused for treating the various conditions described herein, e.g. obesity,fatty liver, diabetes, cancer and the like. In some embodiments of theinvention, the extract is a mushroom extract. The mushroom extract maybe Pleurotus Ostreatus extract. The extract may be prepared as detailedherein.

Dried powder may be prepared from fresh fruiting bodies of PleurotusOstreatus (Yarden) mushrooms following freezing of fresh fruiting bodieswith liquid nitrogen, lyophilizing and afterwards, grinding in in anyappropriate grinder. Alternatively, the mushrooms are frozen at atemperature between −4° C. to −40° C., lyophilized and ground using anyappropriate grinder. In some embodiments the grinding is performed forbetween about 20 seconds to 10 minutes. In some embodiments the grindingis performed for between about 30 seconds to two minutes. In someembodiments the grinding is performed for about one minute. The powderedfruiting bodies may be extracted with water, in some embodiments, coldwater is used by stirring overnight or for a period of between about 20minutes to 6 hours. The extract is then centrifuged at, for example,between about 3,000 rpm to 30 rpm for between about 10 min to two hoursor more. The supernatant is filtered. Aliquots can be tested for Olyexpression or activity in mouse HIB-1B cells.

As shown in Example 7, the freezing of fresh fruiting bodies with liquidnitrogen, followed by lyophiliziation and afterwards grinding providedhigher oly concentration in comparison to mushrooms that were frozen ata temperature of about −20° C., lyophilized and grinded. However, theactivity in mouse HIB-1B cells remained similar in aliquots from bothpreparations. In some embodiments of the invention, additional oly(which may be native or synthetic) may be added to the extract in orderto provide an extract which is enriched by oly.

The following examples are presented in order to more fully illustratecertain embodiments of the invention. They should in no way, however, beconstrued as limiting the broad scope of the invention. One skilled inthe art can readily devise many variations and modifications of theprinciples disclosed herein without departing from the scope of theinvention.

EXAMPLES Experimental Methods Preparation of Ostreolysin

Oly was PCR-amplified using primers containing the NcoI (at the 5′ end)and BamHI+XbaI (at the 3′ end) sites and subcloned to pTrc 99a vector atthe NcoI and XbaI sites. Molecular mass of the protein was 15,400 Da,and the specific absorbance at 280 nm (calculated by DNAman program was2.62 for g/L. The Protein was expressed upon induction with IPTG assoluble protein. The protein was purified by successive extraction,ammonium sulfate precipitation, dialysis, anion exchange chromatography,preparative gel filtration, dialysis and lyophilization in the presenceof NaHCO₃ at approximate protein:salt ration of 1:1. Its concentrationwas calculated according to specific absorbance at 280 nm.

The protein is easily soluble in DDW. Its purity was determined bySDS-PAGE in the presence of reducing agent (see FIG. 1) and byanalytical gel filtration on Supperdex 75 column developed in thepresence of 25 mM Tris-Hcl+300 mM NaCl, Ph 8 (see FIG. 2). The purity asdetermined by both methods was >95% and the molecular mass undernon-denaturative conditions indicate that the protein is a monomer. Inorder to obtain high quantities of the oly, the protein was overexpressed in E. coli and purified (see FIGS. 1 and 2).

Cell Cultures

HIB-1B brown pre-adipocytes [which are derived from a brown fat tumor ofa transgenic mouse, and are the first established brown adipocyte cellline capable of expressing the brown fat-specific mitochondrialuncoupling protein (UCP)] were grown and differentiated or usingrosiglitazone or treating them with Oly. Mouse 3T3-L1 were grown anddifferentiated with rosiglitazone.

RNA Extraction and RT-PCR

Total RNA was isolated using T-Reagent (Sigma). Reverse transcriptionwas performed using High-Capacity cDNA (Applied Biosystems). RT-PCR wasperformed using SYBR Green (Applied Biosystems).

Western Blotting

Primary and secondary antibodies were obtained from Cell SignalingTechnology (Danvers, Mass.) or Santa Cruz Biotechnology (Santa Cruz,Calif.) and western blotting was performed as described inYehuda-Shnaidman, E., et al., Acute stimulation of white adipocyterespiration by PKA-induced lipolysis. Diabetes, 2010. 59(10): p.2474-83.

Immunofluorescence and Confocal Microscopy

Cells were seeded in 12-well plates, covered with glass cover slips andcoated with 0.1% gelatin. A day later, oly (10-62.5 μg/ml) was added for8 h. Cells were fixed in 3.7% (v/v) PFA and permeabilized with 0.5%(v/v) Triton X-100 for three minutes. Cells were incubated with PFA 3.7%for 20 minutes and washed with PBS. After blocking for 1 h at roomtemperature with 5% (v/v) donkey serum, cover slips were incubated withcaveolin-1 primary antibody (dilution 1:100) or oly primary antibody,over night at 40° C. Cover slips were washed with TBST and incubatedwith Alexa Fluor 488 goat anti-rabbit IgG secondary antibody andPhalloidin-TRITC for two hours at room temperature. Additional washeswere performed and finally, mounting solution (70% (v/v)) containingDAPI (30% (v/v)) was added. Cells were observed under confocal ZeissAxiovert 100M microscope (LSM 510, Germany).

Nile Red Staining

Cells were seeded onto 0.1% gelatin coated glass-bottom 24-well culturedishes (MatTak Corporation, Ashland, Mass.). After overnight incubationat 37° C., 5% CO₂, cells were washed with PBS, incubated with 1 μg/mlnile red for 20 minutes at 37° C. and analyzed by confocal microscope.

Example 1 In-Vitro Assay for Assessing the Anti-Proliferative Activityof the Recombinant Oly in HCT-116 Colon Cancer Cell Line

Using a viability assay (MTT assay), the biological anti-proliferativeactivity of the recombinant oly in HCT-116 colon cancer cell line wastested. Similarly to the native protein, the recombinant oly hasanti-proliferative activity (FIG. 3 gray). To further explore whetherthe anti-proliferative activity of oly is specific to cancer cells thatpossess high amounts of lipid drafts, the oly effect on the viability ofnon-cancer cell line, FHS 74 Int (human fetal small intestine), wastested. As shown in FIG. 3 (black column), the anti-proliferativeactivity of oly is much lower in the non-cancer cells (black) vs. thecancer cells (gray).

Example 2 Designation of a Polyclonal Specific Antibody Against theWhole Recombinant Oly

A polyclonal specific antibody against the whole recombinant oly wasdesigned. FIG. 4 demonstrates that the obtained antibody is highlyoly-specific and recognizes both the recombinant and the wild typeprotein.

Example 3 An In-Vitro Assay for Testing the Role of Oly in AdipocyteDifferentiation

Once obtaining an active oly that can penetrate into cells, the oly wastested for its putative role in adipocyte differentiation. Mouse brownpre-adipocyte cell line, HIB-1B, and the mouse white pre-adipocyte cellline, 3T3-L1, were utilized in this test. When HIB-1B cells were treatedwith oly, morphological alterations were observed due to theaccumulation of lipid droplets in the cytoplasm (FIG. 5A). This was alsoevidenced by Nile red staining (FIG. 5B). In contrast, oly-treated3T3-L1 did not show lipid accumulation (FIG. 5B) but affected the geneexpression of some differentiation genes (such as HSL and PGC-1α, notshown). Note: in both cell lines, best effect of oly was detected after24-48 hours. Longer treatment periods did not result in additionalchanges.

To further characterize oly-induced brown adipogenesis, the effect ofoly on gene expression of some adipogenic markers was measured. FIG. 6Apresents that oly induces an increase in the gene expression of aP2,PGC-1α and C/EBPα, while necdin (adipogenic inhibitor) is decreased,suggesting adipocyte differentiation. Moreover, oly increases the geneexpression of specific brown adipogenesis markers, such as: UCP1, CIDEAand prdm16 (FIG. 6B).

It is therefore hypothesized that oly induces differentiation of brownfat and that oly might lead to the transformation of white adipocytesinto ‘brown like’ cells. It is known that white cells can transform intobrown cells.

The involvement of lipid rafts-related proteins in oly-induced brownadipogenesis was also explored. This question is especially relevantbecause of two reasons: (1) oly interacts with lipid rafts that in turnmight lead to its entry through the cell membrane; (2) many accumulatingevidences propose a role for caveolin-1 in adipocyte metabolism.Therefore, the role of caveolin-1 in oly-induced adipogenesis wastested. It was found that oly increases caveolin-1 gene expression inHIB-1B cells (FIG. 7). This suggests a role for caveolin-1 in oly effectand could be part of the differentiation process.

Example 4 In-Vivo Experiment of Toxicity

IP injection to mice of 0.2 mg/kg body weight (BW) or 0.5 mg/kg BW ofrecombinant oly did not induce mortality, nor any sign of sickness ortoxicity in the injected mice, from the application following a weekafter administration.

Example 5 In-Vivo Tests for Assessing the Effect of Oly on Obesity,Diabetes and Fatty Liver Animals and Experiment's Design

Male C57BL/6 mice, 5 weeks old, were purchased from Harlan laboratories,Ein Karem, Jerusalem. All mice were from the same litter. The mice werekept in four plastic cages in the same animal facility, each cagerepresenting a different experimental group, wherein two groups weremaintained on regular diet and two groups received high fat diet (60% offat). Mice were given ad libitum access water. The mice were weighedtwice a week. Following fourteen weeks during which the mice groups wereprovided the two different diet regimes, the injection period with olystarted. The treatment groups (one cage of regular diet and one cage ofhigh fat diet (HFD) mice were injected via the peritoneal cavity with afixed concentration of oly (1.0 μg/gr BW) every other day; each mousewas weighed before the injection, and the injection volume of oly wasadjusted according to the mouse's weight. Control groups were injectedwith similar volumes of saline. In all injections a sterile 1 ml syringewith 26 Ga ⅜″ needle was used. Animal care and experimental procedureswere in accordance with the accredited animal ethics committee of theHebrew University.

Effect of Oly on Body Weight

The weight gain of the four groups of mice during the experiment isshown in FIG. 8, and FIG. 9 presents the weight of the mice on the dayof sacrifice. The results show that oly administration to obese mice,i.e., those provided with a high fat diet, induced significant reductionin body weight.

Effect of Oly on Intraperitoneal Glucose Tolerance Test (IPGTT)

Fasting blood glucose levels were obtained from mice that fasted for 12hours. The test was performed 16 weeks following day 1 of theexperiment. Each mouse was weighed, and fasting glucose levels wereobtained from venous blood from a small tail clip using a glucometer(Optimum Xceed, Abbot, UK) and respective blood glucose test strips(Optimum, Abbot, UK). Afterwards, glucose solution (20% (w/v) in saline)was injected using a 1 ml syringe, 26 Ga ⅜″ needle, according to themouse's weight (2 mg/gr body weight). Blood glucose levels were measuredat 30, 60, 90 and 120 min after the glucose solution injection (see FIG.10A). The area under the curve (AUC) of IPGTT was calculatedrepresenting the body's glucose tolerance for all mice groups (See FIG.10B). As shown in FIGS. 10A and 10B, oly administration to obese miceinduced significant reduction in glucose responsiveness, wherein olysignificantly downregulated glucose intolerance.

Effect of Oly on Food Consumption

The food consumption of the mice throughout the experiment was monitoredand, as shown in FIG. 11, the food consumption of the mice is notaffected by the administration of oly. Accordingly, the evidencedlowering in body weight is not due to lack of appetite or the like.

Effect of Oly on Body Tissues

Following 20 weeks the mice of the different groups were sacrificed anddifferent tissues analyzed as well as the blood samples. The weight ofthe epididymal adipose tissue is presented in FIG. 12. FIG. 13A presentsthe expression of UCP-1, Cidea, PRDM16, perilipin A (brown adipogenicmarkers), which, as shown therein, are upregulated by the administrationof oly. The expression of TNF-α, on the other hand, was downregulated inthe visceral adipose tissue by the administration of oly. It istherefore concluded that the administration of oly induced significantreduction in epididymal fat mass and controlled visceral mass towards amore brown-adipogenic characteristic.

FIG. 14 presents the liver weights of the mice on day of sacrifice,showing that the administration of oly induced a significant reductionin liver mass. Further, the blood was analyzed for liver functions, and,as shown in FIGS. 15 and 16, the transaminases GOT and GPT weresignificantly downregulated by the oly treatment. Further, as shown inFIGS. 17 and 18, the triglyceride and cholesterol levels were alsosignificantly downregulated by the oly treatment.

In addition, the liver samples were tested histologically. As shown inFIG. 19, the livers of the mice provided with a high fat diet proved tobe extremely fatty (see the control HF group), as apparent from therelatively high number of fatty droplets in comparison to the number offatty droplets in the mice provided with a low fat diet. It furtherappears that the oly treatment reduced the fat levels in the liver, asapparent from the normal histological results obtain from the HFD micetreated by oly. The apoptosis in the livers was assessed as well. Aspresented in FIG. 20, oly downregulated the proapoptotic peptide Baxwhile upregulating the antiapoptotic peptide Bcl2, and thereforeBAX/BCL2 was downregulated in liver samples by the administration ofoly, thus inhibiting the death of liver cells. The above results showthat the administration of oly induced significant reduction in fattyliver appearance and associated liver activities.

In addition, various tests were conducted to assess the effect of Oly onNonalcoholic fatty liver disease (NAFLD). The tests and the results arepresented in Table 1 below.

TABLE 1 Gene Expression (Arbitrary Units) Test Explanation Contol Diet(CD) CD + Oly HFD HFD + Oly Transcript Enzyme that generates NADPH 0.48± 0.03 0.47 ± 0.03 0.26 ± 0.01 0.28 ± 0.01 expression used in fatty acidand Statistically of cholesterol biosynthesis. Significant CytosolicPrevious work has correlated malic liver and adipose malic enzyme enzymeexpression with susceptibility to obesity and diabetes. Oly reduces theexpression of this enzyme. Transcript Hepatic insulin resistance 0.18 ±0.02 0.19 ± 0.03 0.10 ± 0.01 0.14 ± 0.02 expression and fatty liver is acritical Statistically of IRS2 component in the development Significantof type 2 diabetes mellitus. Insulin resistance in liver is associatedwith reduced expression of both major insulin receptor substrate (IRS)proteins, IRS-1 and IRS-2. Oly sensitizes the liver to insulin byupregulating IRS2. Transcript Fatty acid synthase (FASN) 0.10 ± 0.050.08 ± 0.02 0.14 ± 0.01 0.11 ± 0.02 expression catalyzes the last stepin Statistically of FASN fatty acid biosynthesis, and Significant is amajor determinant of the maximal hepatic capacity to generate fattyacids by de novo lipogenesis. FASN mRNA expression in human control vsNAFLD livers confirmed significantly higher FASN levels in hepaticsteatosis. Oly downregulates FASN expression. Transcript Tissue-specificoverexpression of 0.12 ± 0.03 0.08 ± 0.02 0.14 ± 0.01 0.125 ± 0.02 expression lipoprotein lipase (LPL) causes Statistically of LPLtissue-specific insulin Significant resistance. It happens in fattyliver. Oly inhibits expression of this enzyme. Transcript PKLR is theprotein encoded by 0.14 ± 0.04 0.08 ± 0.01 0.19 ± 0.02 0.09 ± 0.01expression the gene pyruvate kinase Statistically of PKLR that catalyzesthe Significant transphosphorylation of phohsphoenolpyruvate intopyruvate and ATP, which is the rate-limiting step of glycolysis. Itprovides the substrate for glycerol and fatty acid accumulation in theliver. Oly inhibits expression of this enzyme. Transcript IKKE and TBK1are part of  0.01 ± 0.001  0.01 ± 0.002 0.035 ± 0.003  0.01 ± 0.001expression a process of inflammation Statistically of IKKE linked toobesity and Significant insulin resistance, the condition that precedesType 2 diabetes. Oly downregulates them. Transcript IKKE and TBK1 arepart 0.09 ± 0.01 0.07 ± 0.02 0.11 ± 0.01 0.06 ± 0.01 expression of aprocess of Statistically of TBK1 inflammation linked to Significantobesity and insulin resistance, the condition that precedes Type 2diabetes. Oly downregulates them. Transcript CCL-2 or MCP-1 play a 0.04± 0.01 0.05 ± 0.01 0.19 ± 0.02 0.06 ± 0.01 expression significant rolein hepatic Statistically of CCL2 or steatosis or early liver SignificantMCP-1 injury. Transcript CCL3 is elevated in the  0.9 ± 0.1 ND 2.4 ± 0.3 0.4 ± 0.05 expression plasma and metabolic Statistically of CCL3tissues (liver and adipose Significant tissue) of patients withhyperlipidemia and metabolic disease. Thus CCL3 is an importantchemokine in recruitment of immune cells to metabolic tissues. Olydownregulates its expression Transcript EMR1 or EGF-like module 0.041 ±0.01  0.041 ± 0.01  0.05 ± 0.02 0.038 ± 0.01  expression containingmucin-like Statistically of EMR1 hormone receptor-like 1 Significant[EMR1] is a marker of inflammation in the liver, downregulated by Oly

Example 6 Effect of Oly in Cancer Methods Cell Lines and CultureConditions

HCT116 colorectal carcinoma cells (ATCC number: CCL-247) were maintainedin Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich, Israel)supplemented with 10% (v/v) Fetal bovine serum (FBS; BiologicalIndustries, Beit Haemek, Israel) and 0.2% (v/v)penicillin-streptomycin-nystatin. HM7 highly metastatic colon cancercells from clone #1 (HM7 cells transfected with pcDNA3 neo plasmid, notexpressing Caveolin-1) and clone #15 (HM7 cells transfected withpcDNA-Caveolin-1, Plasmid with Caveolin-1 protein insert, expressinghigh levels of Caveolin-1) were maintained in DMEM supplemented with 10%(v/v) FBS and 0.275% (v/v) G-418 (Gibco, Paisley, UK). All cells werecultured in 5% CO₂ in a humidified atmosphere at 37° C.

Anti-Cancer Activity (MTT Assay)

HCT116 cells and HM7 cells from clone #1 and clone #15 were seeded in96-well plate (2.0×10⁴ per well). After pre incubation over night at 37°C. in a CO₂ incubator, the recombinant protein Ostreolysin, prepared byexpression in E. coli, was added to cell cultures at a concentration of125 μg/ml and 62.5 μg/ml. Fresh medium alone was added to the control.Fruiting body extract was added at three concentrations: 0.01% (w/v),0.025% (w/v), 0.05% (w/v). After incubation for 4, 8, 12, 24 hours at37° C., medium was removed and 50 μl of3-(4-,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazoliumbromide (MTT)solution (0.5 mg/ml) was added. The plates were incubated for 1 hr at37° C. After MTT solution was removed, 100 μl DMSO were added to eachwell and the plates were shaken for 20 min. Formation of coloredformazan was assessed at 550 nm in ELx 808 Ultra microplate reader(BIO-TEK INSTRUMENTS INC) using KC junior software.

Cell Cycle Analysis

HCT116 cells were plated in 6-well plates (9.0×10⁵ cells per well) andallowed to adhere overnight. Fresh medium was added to the cell culturewith the recombinant protein Ostreolysin at a concentration of 125μg/ml. Fresh medium alone was added to the control. Fruiting bodyextract was added at a concentration of 0.01% (w/v). After 8 hoursincubation, the cells were washed in PBS, trypsinized, harvested andre-suspended in 0.5 ml sterile PBS. 0.5 ml of cold 70% (v/v) ethanol wasadded to cell suspensions while vortexing and the samples were stored at4° C. For staining, cells were centrifuged for 5 min at 1500 rpm(Hettich Zentrifugen Rotofix 32), the upper layer was discarded and DNAfragmentation solution (0.05 mg/ml propidium iodide, 0.1% (v/v) TritonX-100 and 0.1% (w/v) sodium citrate) was added for 1 hour incubation onice. The DNA content was measured by exciting propidium iodide at 488 nmand measuring the emission at 575 nm (FL2) using a flow cytometer (BDFACScalibur BD Biosciences, San Jose, Calif.). Analysis was performed byWinMDI 2.9 software.

Western Blotting and Densitometry

HCT116 cell lysates were, electrophoresed in 12% SDS-PAGE, transferredto nitrocellulose transfer membranes (Whatman, Schleicher, Schuell,Dassel, Germany), blocked in TBST containing 5% (w/v) dry nonfat milkand incubated with PARP-1 (dilution 1:1000) or BAX (1:500) or β-actin(dilution 1:10,000) antibody over night at 4° C. Membranes weresubsequently incubated with a secondary anti-rabbit antibody coupled tohorseradish peroxidase (Jackson I R, Baltimore, Pa., USA, dilution1:10,000) for 1 hour at room temperature. Proteins were visualized usingan ECL kit. Effective transfer to nitrocellulose membrane was confirmedby staining with Ponceau S. Films were scanned by a Mustek 1200 UB Plusscanner (Mustek systems Inc., CA, USA). Densitometry was assessed usingthe Gelpro32 analyzer software and β-actin was used as a loadingcontrol.

Immunofluorescence and Confocal Microscopy

HCT116 cells were seeded at a density of 3.6×10⁵ cells per well on glasscover slips (diameter, 1.8 cm) coated with 0.1% gelatin, placed in12-well plates. The cells were allowed to adhere over night andtreatments were applied as follows: Fresh medium was added to cellculture with the recombinant protein oly at a concentration of 125μg/ml. Fresh medium alone was added to the control. Fruiting bodyextract was added at a concentration of 0.01% (w/v). After 4 or 8 hoursincubation, the cells were fixed with 3.7% (v/v) PFA and permeabilizedwith 0.5% (v/v) Triton X-100 for 3 min. Afterwards, the cells wereincubated with PFA 3.7% for 20 min and washed three times with PBS. Toblock unspecific staining, the cells were incubated for 1 hr at roomtemperature with 5% (v/v) donkey serum in TBST. The cells weresequentially stained with Caveolin-1 primary antibody (dilution 1:100)or Ostreolysin primary antibody (dilution 1:500) or Flotillin-1 primaryantibody (dilution 1:100) in a humidity chamber over night at 4° C.Cover slips were washed three times for 30 min with TBST and thenincubated with Alexa Fluor 488 goat anti-rabbit IgG secondary antibody(dilution 1:500) and Phalloidin-TRITC in a humidity chamber for twohours at room temperature. Cover slips were washed four times with TBSTand then mounted upside-down with mounting solution (70% (v/v)) mixedwith mounting solution with DAPI (30% (v/v)) on glass slides. The cellswere observed under Leica CTR4000 Confocal microscope (Mannheim,Germany) at ×63 magnification using immersion oil.

Results Recombinant Ostreolysin Exerts an Anti Proliferative Effect onHCT116 Cells and HM7 Clones

Exposure of HCT116 (colorectal carcinoma cells) to oly has showncytotoxicity with an effective concentration of 125 μg/ml producing a50% decrease in cell viability at 8 hours (FIG. 21C). The cytotoxiceffect of oly was also observed in HM7 (highly metastatic colon cancer).HM7 clone #15 (expressing high Cav-1 levels) presented a significantincreased sensitivity to oly as compared with clone #1 (no Cav-1expression) when administered for 4 and 8 hours (FIGS. 21B and 21D,respectively).

Direct microscope observation of cell morphology confirmed that oly hada similar effect on HCT116 cell line and HM7 clones, producing shrinkageof cells (data not shown). The cytotoxic activity of oly had alreadypeaked after 4 hours of incubation in both cell lines.

In order to test whether the cytotoxic effect of oly is specific tocancer cells, the oly effect on the viability of non-cancer cell line,FHS 74 Int (human fetal small intestine) was also tested. As seen inFIG. 3 (see in Example 1), the anti-proliferative activity of oly ismuch lower in the normal cells (FHS 74 Int, black) vs. the cancer cells(HCT116, gray), implying a specific anti-proliferative role for oly incancer cells.

Cell Cycle Analysis of HCT116 Cell Line Indicated that Recombinant OlyInduces Apoptosis in These cells

In order to quantify cell distribution in apoptosis and cell cycle,HCT116 cell line was analyzed by flow cytometry (FIG. 22A) withouttreatment (control) or with oly 125 μg/ml and FBE 0.01% (w/v). Afterstaining with a quantitative DNA-binding dye, cells that have lost DNAvia apoptosis will take up less stain and will appear as a sub-G1 peakto the left of the G1 peak. The results demonstrate that in HCT116untreated cells, cell cycle distribution was 1.238±0.124, 42.482±1.709,13.198±0.845, 27.997±0.856 in apoptosis, G0/G1, S, G2/M respectively. InHCT116 cells treated with FBE 0.01% (w/v) cell cycle distribution was2.045±0.326, 33.807±1.109, 11.483±0.726, 27.560±1.102 in apoptosis,G0/G1, S, G2/M respectively. In HCT116 cells treated with oly 125 μg/mlcell cycle distribution was 7.380±0.584, 46.048±2.307, 13.022±1.158,25.988±0.487 in apoptosis, G0/G1, S, G2/M respectively. The differencesbetween untreated HCT116 cells and oly 125 μg/ml treated HCT-116 cellswere significant with P-value of <0.05 (FIG. 22B).

Recombinant Ostreolysin Promotes the Cleavage of PARP-1 and Expressionof BAX Pro-Apoptotic Markers in HCT116 Cell Line (Colon Cancer HumanCell Line)

The extent of apoptosis was assessed by detection of active PARP-1 anddetection of BAX protein using western blot. PARP, a 116 kD nuclear poly(ADP-ribose) polymerase is one of the main cleavage targets of caspase-3in-vivo. Cleavage separates the PARP amino-terminal DNA binding domain(24 kD) from the carboxy-terminal catalytic domain (89 kD) (10) andserves as a marker of cells undergoing apoptosis. In HCT116 cell line,oly 125 μg/ml treatment induced cleavage of PARP (FIG. 23A), revealed byan antibody that recognizes both the full-length 116 kD fragment as wellas the 89 kD cleaved fragment. BAX is a 23 kD pro-apoptotic protein,member of the Bcl-2 family. The critical events in the activationprocess of BAX are its translocation to mitochondria and its N-terminalconformational change closely coupled to mitochondrial membraneinsertion and oligomerisation. The insertion of BAX into themitochondrial outer membrane is closely associated with the release intothe cytosol of several proteins such as cytochrome c and procaspase-3which are essential to the execution of the apoptotic program. BAXactivation was investigated by Western blot using an antibody thatspecifically recognizes the activated conformation of BAX. Oly 125 μg/mltreatment of HCT116 cells induced an increase in activated-BAX ascompared to non-treated conditions (FIG. 23B). Quantification of thenumber of BAX positive apoptotic cells revealed that oly significantlyaffects overall apoptosis as compared with control untreated cells (FIG.23C).

Recombinant Ostreolysin Interacts with the Cell Membrane and Enters theCytosol in HCT116 Cells.

As previously reported, selective binding and clustering of oly onchondrocyte membranes, combined with results obtained from artificialmembranes and Chinese hamster ovary cells, indicate that thedistribution of oly molecules bound to the membranes was not uniformlydistributed over the cell surface, but was concentrated in many focalclusters. This suggests that oly recognizes distinct membrane domainsthat probably serve as attachment sites for aegerolysin-like proteinsleading to their aggregation and formation of the pore.

Next, the membrane distribution of recombinant oly after oly and FBEtreatments of HCT116 cells as compared with control conditions, (FIG.24) were investigated. Cells treated for 8 hours with recombinant oly125 μg/ml presented a greater distribution of oly-rich domains ascompared to control and FBE conditions (FIG. 24). In addition, crosssection images of oly treated cells demonstrated that the recombinantoly penetrates the cell membrane and enters the cytosol.

Recombinant Oly Induces Reorganization and Clustering of Cav-1-RichMembrane in HCT116 Cells.

It is thought that upon extracellular stimulus, the plasma membrane isprepared for the formation of more stabilized domains and molecularclusters with enhanced size and lifetime such as Caveolae. In order tounderstand the involvement of Caveolin-1 in apoptosis of colon cancercells, the effect of oly stimulation on HCT116 cell line was explored(FIG. 25). Caveolae are evident as circular profiles with uniform shapeand 50-100 nm in diameter, which are formed by the polymerisation ofcaveolins leading to the clustering and invagination of existingcholesterol sphingolipid rich domains (lipid rafts) in the cell plasmamembrane. Therefore, the membrane distribution of Cav-1 in controlconditions and after oly treatment was investigated. Cells treated for 8hours with oly 125 μg/ml presented a greater number of Cav-1-richdomains as compared to control conditions (FIG. 25). In contrast (FIG.26) Oly did not induced any notable up-regulation of expression of thelipid-raft associated protein Flotilin-1.

In-Vivo Anticancer Experiments

C57Bl mice were subcutaneously inoculated with an MC38 colon carcinomacell line, resulting in development of in situ of very aggressive tumorswithin 3 weeks after cell inoculation. FIG. 27 demonstrates the effectof Oly on MC38-derived colon cancer cells implanted in C57Bl mice. Thecells were injected subcutaneously (2×10⁵ cells per mouse) into the lefthip. After 10 days of injection and following appearance of tumor signsin some mice, the mice were treated with 1 mg/kg Oly, (3 times a weekintraperitoneally). Control mice received PBS 3 times a weekintraperitoneally. Each bar represents the standard error of the mean.N=8 mice; mice were sacrificed on day 39. *=P<0.001. As can be seen olysignificantly reduced the size of the tumor. FIG. 28 demonstrates thebeneficial effect of oly on the tumor weight (which is about half thesize of the control).

Example 7 Preparation of Pleurotus Ostreatus Extracts PleurotusOstreatus Preparation Method 1:

Dried powder was prepared from fresh fruiting bodies of PleurotusOstreatus (Yarden) mushrooms following freezing of fresh fruiting bodieswith liquid nitrogen, lyophilizing and afterwards grinding for 1 min.Each 10 gram of powdered fruiting bodies were extracted with 100 ml ofcold water (4° C.) by stirring overnight and the mixture was centrifugedat 10,000 rpm for 30 min. The supernatant was filtered, aliquots testedfor Oly expression by Western Blot (in the future ELISA methodology totest Oly concentration will be developed) and 10 microliter aliquotswere used in order to test the activity in mouse HIB-1B cells.

Pleurotus Ostreatus Preparation Method 2:

Dried powder was prepared from fresh fruiting bodies of PleurotusOstreatus (Yarden) mushrooms by freezing the samples at −20° C.,lyophilized and grinding in Moulinex for 1 min. Each 10 gram of powderedfruiting bodies was extracted with 100 ml of cold (4° C.) water bystirring overnight and centrifuged at 10,000 rpm for 30 min. Thesupernatant was filtered, aliquots tested for Oly expression by WesternBlot and 10 microliter aliquots were used in order to test the activityin mouse HIB-1B cells.

Comparison of Oly Concentration in the Two Pleurotus OstreatusPreparations

As can be seen from FIG. 29 which show the Western blot analysis Olyconcentration in extracts from powdered Pleurotus Ostreatus afterfreezing in liquid nitrogen (method 1) are higher than in extracts frompowdered Pleurotus Ostreatus after freezing at −20° C. (method 2).

Biological Test of the Two Pleurotus Ostreatus Preparations

The samples were tested biologically.

The biological test used was the appearance of round bodies resemblingintracellular lipid droplets inside HIB-1B cells, which are derived froma brown fat tumor of a transgenic mouse, and are the first establishedbrown adipocyte cell line capable of expressing the brown fat-specificmitochondrial uncoupling protein (UCP).

HIB-1B cells were exposed to 10 μl of a dilution of 1/10 preparation(dilution in distilled sterile water) of Pleurotus Ostreatus preparationMethod 1 or a similar amount of Pleurotus Ostreatus preparation Method2.

Results

(It is noted that for 24 h incubation, similar results were obtained for48 h incubation; however, the data is not shown)

As can be seen from FIG. 30, in the control HIB-1B cells no lipiddroplets were seen (FIG. 31A). In the Oly (10 μg/ml) treated HIB-1Bcells many lipid droplets can be seen (FIG. 31B). In the PleurotusOstreatus preparation Method 1-treated HIB-1B cells.

HIB-1B cells treated with fruiting bodies extract from PleurotusOstreatus according to Method1. Lipid droplets were seen in an amountwhich was similar to that of the oly treated cells (FIG. 31C).Similarly, as can be seen from FIG. 31D, which show the PleurotusOstreatus preparation Method 2-treated HIB-1B cells, the amount of lipiddroplets in the cells was similar to the amount of the droplets in theoly treatment.

In conclusion both methods of preparation of the extracts resulted in asufficient Oly-like activity. Even at lower concentrations of oly, thepreparations are effective. Therefore, it is expected that in-vivoadministration of these Pleurotus Ostreatus mushroom

isolates will induce as anti-obesity, anti-insulin resistance,anti-cancer and anti-fatty liver effects as was shown for therecombinant oly.

It will be appreciated by persons skilled in the art that the presentinvention is not limited by what has been particularly shown anddescribed herein above. Rather the scope of the invention is defined bythe claims that follow.

1. A method of treating obesity or overweight comprising the step ofadministering to a subject in need a formulation comprising atherapeutically effective amount of ostreolysin, or a functionallyrelated variant of ostreolysin having at least 95% sequence identity toSEQ ID NO:
 1. 2. The method of claim 1, wherein the functionally relatedvariant of ostreolysin has at least 99% sequence identity to SEQ IDNO:1.
 3. The method of claim 1, wherein the formulation is apharmaceutical formulation.
 4. The method of claim 1, wherein theformulation is in the form of a powder, solution, enteric coated table,suspension, emulsion, tablet, capsule, enteric coated tablet, gel,cream, ointment, foam, paste or injection.
 5. The method of claim 1,wherein the ostreolysin is a recombinant protein.
 6. The method of claim1, wherein the ostreolysin is a purified protein.
 7. The method of claim1, wherein the therapeutically effective amount of ostreolysin isbetween 5-10 mg for a 60 kg individual.
 8. A method of enhancingproduction of brown fat adipocytes, or promoting brown adipocytedifferentiation, the method comprising contacting a precursor of brownfat adipocytes with a composition comprising an effective amount ofostreolysin, or a functionally related variant of ostreolysin having atleast 95% sequence identity to SEQ ID NO: 1, so as to enhance productionof brown fat adipocytes or promote brown adipocyte differentiation. 9.The method of claim 7, wherein the functionally related variant ofostreolysin has at least 99% sequence identity to SEQ ID NO:1.
 10. Themethod of claim 7, wherein the enhancing of production of brown fatadipocytes or promoting brown adipocyte differentiation is effective intreating overweight or obesity in a subject in need thereof.
 11. Themethod of claim 7, wherein the composition is in a form of a powder,solution, suspension, emulsion, tablet, enteric coated tablet, orcapsule, gel, cream, ointment, foam, paste or injection.
 12. The methodof claim 7, wherein the precursor of brown fat adipocytes is a stemcell, mesenchymal stem cell, myogenic precursor, brown pre-adipocyte andwhite pre-adipocyte.
 13. The method of claim 7, wherein the formulationis a pharmaceutical formulation.
 14. The method of claim 7, wherein theostreolysin is a recombinant protein.
 15. The method of claim 7, whereinthe ostreolysin is a purified protein.
 16. The method of claim 7,wherein the therapeutically effective amount of OLY is between 5-10 mgfor a 60 kg individual.